Project Summary Aminoacyl-phosphatidylglycerol synthases (aaPGSs) are proteins responsible for the synthesis of aminoacyl-phosphatidylglycerol (aa-PG) in the bacterial membrane. To synthesize aa-PG, these enzymes transfer the amino acid (aa) of aminoacylated transfer ribonucleic acids (aa- tRNAs) onto the free hydroxyl group of PG. This modification mechanism is found in a wide variety of bacteria, and increases the adaptability of these organisms to a range of challenging environmental conditions such as low pH and osmotic stress. In pathogens, such as Staphylococcus aureus and Mycobacterium tuberculosis, aminoacylation of PG helps the organism evade the immune system of the host and resist several classes of antibiotics. aa-PGs confer this protection by altering the charge of the cellular membrane, and by modulating more general biophysical properties such as membrane fluidity and permeability. Because these proteins are involved in virulence and antibiotic resistance, and because they are present in many bacterial species, they represent attractive targets for therapeutic strategies to combat infection. Basic insight into the fundamental processes underlying aa-PG formation is needed to develop future therapeutical strategies. For instance, very little is known about how aaPGSs recognize their substrates and carry out the membrane modification reaction. Also, the antimicrobial resistances associated with aa-PG synthesis have been described only for a few pathogens, and almost nothing is known about the regulatory control of this pathway. During the course of this proposal we will investigate the molecular mechanism used by aaPGSs to recognize aa-tRNAs and catalyze formation of aa-PG. This portion of the study will be conducted using two proteins from Clostridium perfringens, which utilize two distinct amino acids (Ala, and Lys) for membrane modification, representing the two main types of aaPGSs described to date. The second goal of this proposal is to determine the antibiotic resistances associated with the aa-PG response in Enterococcus faecium, which is an emerging nocosomial pathogen. Regulation of the aa-PG response in E. faecium, and the suitability of other physiologically relevant amino acid donors (i.e, Ala-tmRNA) as substrates for aaPGS activity, will be investigated in vivo and in vitro.